The highly conserved p7 nucleocapsid protein (NCp7) of HIV-1 is a target for the development of new antiviral agents based on its broad range of function in virus replication. NCp7 plays pivotal roles during both early and late phases of HIV-1 replication, being required for the functioning of reverse transcriptase, integrase and protease, as well as the selection and packaging of the RNA genome into mature virions. Mutations in the Zn chelating and/or non-chelating residues have been shown to result in loss of NCp7-mediated functions, rendering the virus non-infectious. NCp7 contains two copies of the zinc finger motif Cys(X)2Cys(X)4His(X)4Cys(CCHC). Diverse sets of electrophilic compounds that react with the cysteine thiolates in the NCp7 protein or NCp7 protein precursors (p55gag and pr160gag-pol) have been identified. Although different in chemical composition, all lead molecules cause the ejection of Zn (II) ions bound within the structural Zn finger motifs of the NC protein. As a result of our efforts to increase solubility, stability and potency we have recently identified a series of less uncharged S-acyl 2-meraptobenzamide thioester derivatives. In order to maximize the potential to identify the optimal compound configuration that elicits antiviral activity, we developed a combinatorial chemistry approach that allowed the use of substituents which could potentially modify the reactivity of the thioester bond through electronic influences and steric hindrance. With this approach, we tested the hypothesis that by increasing stability of the thioester linkage we would enhance the potency of the thioester chemotype and achieve sub-micromolar inhibitory efficacy. Our data revealed that there was no significant correlation between thioester stability and antiviral activity; however, a slight inverse correlation between stability in serum and virucidal activity was observed. Based on this virucidal capability and our ability to select lead compounds to inhibit virus expression from latenly infected TNF-alpha-induced U1 cells, we tested these compounds to determine whether they could prevent HIV cell-to-cell transmission. Seven thioesters demonstrated potent inhibition of cell-to-cell transmission in the 80 to 100 nanomolar range. Thus, these selected compounds show important potential for development as topical microbicides. These compounds are active against HIV-1, HIV-2 and simian immunodeficiency virus (SIV), indicating a highly conserved target. To study the mechanism of action of our lead compounds, we used both in vitro and in vivo methods. A radiolabeled thioester was studied for its action on HIV-infected human cells in culture. Visualization of electrophoretic patterns of intracellular proteins by autoradiography and by Western blotting with antibody specific for NCp7 revealed clearly that NCp7 is indeed a target for the drug and that it is inactivated by an acylation reaction from a portion of the thioester. Additional biochemical experiments and NMR structural studies have revealed that acyl adducts form at several cysteines on the distal zinc finger of NCp7 from the action of our thioesters on this target. These candidate drugs are being used to study further the structural and functional roles of NCp7 in various stages of the replication cycle, including the intact virion. Further studies of the action of the newer NCp7 inhibitors will shed light on organ and cellular distribution of the virus. These studies involve the kinetics of inhibition of virus replication and infectivity by a covalent drug that irreversibly inactivates a core structure of HIV both intra- and extracellularly. Targeting the highly conserved nucleocapsid protein of HIV should greatly enhance success in finding a drug that can irreversibly inactivate drug-resistant strains of HIV without giving rise to its own drug resistance and also be active against other retroviruses.